Assessment of simple marker-free genetic transformation techniques in alfalfa

The Asteraceae family is the largest and most diversified family of the Angiosperms, characterized by the presence of numerous clustered inflorescences, which have the appearance of a single compound flower. It is estimated that this family represents around 10% of all flowered species, with a great biodiversity, covering all environments on the planet, except Antarctica. Also, it includes economically important crops, such as lettuce, sunflower, and chrysanthemum; wild flowers; herbs, and several species that produce molecules with pharmacological properties. Nevertheless, the biotechnological improvement of this family is limited to a few species and their genetic transformation was achieved later than in other plant families. Lettuce (Lactuca sativa L.) is a model species in molecular biology and plant biotechnology that has easily adapted to tissue culture, with efficient shoot regeneration from different tissues, organs, cells, and protoplasts. Due to this plasticity, it was possible to obtain transgenic plants tolerant to biotic or abiotic stresses as well as for the production of commercially interesting molecules (molecular farming). These advances, together with the complete sequencing of lettuce genome allowed the rapid adoption of gene editing using the CRISPR system. On the other hand, sunflower (Helianthus annuus L.) is a species that for years was considered recalcitrant to in vitro culture. Although this difficulty was overcome and some publications were made on sunflower genetic transformation, until now there is no transgenic variety commercialized or authorized for cultivation. In this article, we review similarities (such as avoiding the utilization of the CaMV35S promoter in transformation vectors) and differences (such as transformation efficiency) in the state of the art of genetic transformation techniques performed in these two species.

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Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

Frontiers in Plant Science ◽

10.3389/fpls.2021.768197 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gabriela Conti ◽

Beatriz Xoconostle-Cázares ◽

Gabriel Marcelino-Pérez ◽

Horacio Esteban Hopp ◽

Carina A. Reyes

Keyword(s):

Genetic Transformation ◽

New Technologies ◽

Negative Impact ◽

Protoplast Isolation ◽

Future Perspective ◽

Juvenile Period ◽

Breeding Programs ◽

Regulatory Processes ◽

Marker Free

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

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PENGARUH JENIS AUKSIN TERHADAP PEMBENTUKAN KALUS DAN DAYA REGENERASI TIGA VARIETAS PADI LOKAL

Berkala Ilmiah Pertanian ◽

10.19184/bip.v2i2.16175 ◽

2019 ◽

Vol 2 (2) ◽

pp. 77

Author(s):

Zaiyin Rizky Ageng Maulidia ◽

Wahyu Indra Duwi Fanata

Keyword(s):

Genetic Diversity ◽

Experimental Design ◽

Plant Regeneration ◽

Genetic Transformation ◽

Callus Induction ◽

Plant Tissue ◽

Transformation Techniques ◽

Rice Varieties ◽

Rice Genetic

ABSTRACT Indonesia is rich in rice genetic diversity in the form of javanica rice. Subspecies javanica have some superiority characters, among others a strong plant tissue, but in generally they have low productivity. Weaknesses of javanica rice can be repaired by biotechnology through genetic transformation techniques from callus of plant. Auxin types and genotypes are important factors for callus induction and plant regeneration. Therefore, this research held for know callus introduction and plant regeneration of three varieties of Javanica rice (Pendok, Genjah Arum, and Menthik Wangi Susu) combined with type of auxin (2,4-D and pychloram). The experimental design of RALs two factorials, there are three varieties of rice (Pendok, Genjah Arum, and Menthik Wangi Susu) and two types of auxin (2,4-D and Pychloram) and analyzed by DMRT α5%. The results of this study is Mentik Wangi Susu have highest regeneration than Pendok and Genjah Arum. The use of 2,4-D resulted in a higher callus induction and plant regeneration than pychloram in the three rice varieties used. While the best combination of treatments for callus induction and regeneration is Menthik Wangi Susu and 2,4-D. Keyword: auxin, javanica, callus induction, plant regeneration. ABSTRAK Indonesia kaya akan keragaman genetik padi berupa padi javanica. Padi sub spesies javanica memiliki beberapa keunggulan diantaranya jaringan tanaman yang kuat, namun pada umumnya mempunyai tingkat produktivitas gabah yang rendah. Kelemahan pada padi javanica dapat diperbaiki dengan bioteknologi melalui teknik transformasi genetik menggunakan jaringan kalus. Genotip dan jenis auksin merupakan faktor penting dalam induksi kalus dan regenerasi tanaman padi secara in vitro. Oleh karena itu, penelitian ini dilaksanakan untuk mengetahui daya pembentukan kalus dan regenerasi pada tiga varietas padi javanica, yaitu Pendok, Genjah Arum, dan Menthik Wangi Susu yang dikombinasikan dengan perlakuan jenis auksin berupa penggunaan 2,4-D dan pikloram. Rancangan percobaan yang digunakan adalah RAL dua faktorial yaitu varietas tanaman padi dan jenis auksin dengan pengujian menggunakan DMRT α5%. Hasil penelitian menunjukkan bahwa dari ketiga varietas padi yang digunakan, varietas Mentik Wangi Susu menunjukan daya regenerasi yang tertinggi dibandingkan varietas Pendok dan Genjah Arum. Penggunaan 2,4-D menghasilkan tingkat pengkalusan dan regenerasi yang tinggi dibandingkan pikloram pada tiga varietas padi yang digunakan. Sedangkan kombinasi perlakuan yang terbaik untuk induksi kalus dan daya regenerasi adalah penggunaan varietas Menthik Wangi Susu dan auksin dalam bentuk 2,4-D. Kata Kunci: auksin, javanica, induksi kalus, regenerasi tanaman.

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Genetic Transformation Techniques in Cryptanalysis

Intelligent Information and Database Systems - Lecture Notes in Computer Science ◽

10.1007/978-3-319-05458-2_16 ◽

2014 ◽

pp. 147-156 ◽

Cited By ~ 9

Author(s):

Urszula Boryczka ◽

Kamil Dworak

Keyword(s):

Genetic Transformation ◽

Transformation Techniques

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Induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes

Open Life Sciences ◽

10.2478/s11535-013-0157-6 ◽

2013 ◽

Vol 8 (5) ◽

pp. 470-479 ◽

Cited By ~ 3

Author(s):

Mariya Petrova ◽

Ely Zayova ◽

Mariana Vlahova

Keyword(s):

Genetic Transformation ◽

Agrobacterium Rhizogenes ◽

Hairy Roots ◽

Culture Media ◽

Pcr Analysis ◽

Transformed Roots ◽

Fresh Weight ◽

Transformation Techniques ◽

Nutrient Media ◽

Arnica Montana

AbstractThe induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes mediated system was established. The frequency of genetic transformation varied from 4.8 to 12% depended on method of infection. The cefotaxime at concentration of 200 mg/l proved to suppress effectively the growth of A. rhizogenes after co-cultivation. Among the three tested nutrient media: Murashige and Skoog (MS), Gamborg’s (B5) and Schenk and Hildebrandt (SH), MS medium was superior for growth and high biomass production of transformed roots compared to other culture media. After culturing for 40 days the fresh weight of clone T4 increased 7.6 fold over the non-transformed roots. The transfer of rol A, rol B and rol C genes into Arnica genome was confirmed by PCR analysis. Established genetic transformation techniques in A. montana efficiently provided and generated a large number of transformed roots — an excellent system for studying gene function and could be used for the production of secondary metabolites synthesized in roots.

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In Vitro Propagation of Chrysanthemum: an Overview on its Utility in Mutagenesis and Genetic Transformation Techniques

Agricultural Research & Technology Open Access Journal ◽

10.19080/artoaj.2018.15.555962 ◽

2018 ◽

Vol 15 (4) ◽

Author(s):

Fardin Nasri

Keyword(s):

Genetic Transformation ◽

In Vitro Propagation ◽

Transformation Techniques

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Progress in Tissue Culture and Genetic Transformation of Oil Palm: An Overview

International Journal of Molecular Sciences ◽

10.3390/ijms20215353 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5353 ◽

Cited By ~ 4

Author(s):

Rajesh Yarra ◽

Longfei Jin ◽

Zhihao Zhao ◽

Hongxing Cao

Keyword(s):

Tissue Culture ◽

Genetic Transformation ◽

Oil Palm ◽

Large Scale ◽

Elaeis Guineensis ◽

Microprojectile Bombardment ◽

Oil Consumption ◽

Transformation Techniques ◽

Significant Research

Oil palm (Elaeis guineensis, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, Agrobacterium tumefaciens mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.

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In vitro organogenesis optimization and plantlet regeneration in Citrus sinensis and C. limonia

Scientia Agricola ◽

10.1590/s0103-90162002000100004 ◽

2002 ◽

Vol 59 (1) ◽

pp. 35-40 ◽

Cited By ~ 17

Author(s):

Weliton Antonio Bastos de Almeida ◽

Francisco de Assis Alves Mourão Filho ◽

Beatriz Madalena Januzzi Mendes ◽

Adriana Pinheiro Martinelli Rodriguez

Keyword(s):

Genetic Transformation ◽

Citrus Sinensis ◽

Sweet Orange ◽

Adventitious Bud ◽

Transformation Techniques ◽

Rangpur Lime ◽

Bud Induction ◽

Bud Growth ◽

Exogenous Genes

Exogenous genes can be introduced in plants by genetic transformation techniques. However, an efficient tissue culture system with high rates of plant recovery is necessary for gene introduction. This work aimed to define organogenesis and plant regeneration protocols for sweet orange varieties Natal, Valencia and Hamlin (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck) which can be used in plant transformation experiments. Seeds of which teguments were removed, were germinated in vitro and maintained in the dark for three weeks, followed by one week at 16-h photoperiod (40 µmol m-2 s-1) and 27 ± 2°C. Organogenesis induction was done by introducing epicotyl segments in MT medium with 25 g L-1 sucrose and different BAP concentrations. After adventitious bud growth, the shoots were transferred to MT medium with either NAA or IBA (1 mg L-1), or absence of auxin, for rooting. The best results were obtained with 1 mg L-1 BAP for bud induction and 1 mg L-1 IBA for rooting for all three sweet orange cultivars. The use of 0.5-2.5 mg L-1 BAP, followed by 1 mg L-1 IBA were the best growth regulator combinations for bud induction and rooting, respectively, for 'Rangpur' lime. The protocols presented in this work are suitable for associations with genetic transformation experiments for these cultivars.

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